The introduction of highly stable Extended-Spectrum cephalosporins at the beginning of the 1980's was quickly followed by the emergence of several cephalosporin resistant Extended-Spectrum beta-lactamases identified among clinical isolates of Klebsiella pneumoniae. Homology studies of these enzymes suggested that bacterial have adapted to the antibiotics by altering (amino acid substitutions) existing plasmid-mediated beta-lactamases such to expand their spectrum of activity. Subsequently, plasmids encoding altered beta-lactamases were disseminated among gram-negative clinical isolates. This resulted in increased beta-lactam antibiotic resistance among clinical gram-negative bacteria. In this proposal, an extensive investigation will be conducted to identify and characterize novel beta-lactamases (derrived from amino acid substitutions) identified in clinical isolates of Klebsiella pneumoniae. Cephalosporin resistant Klebsiella pneumonia will be identified by Kirby-Bauer disk diffusion susceptibility testing using disk impregnated with cephalosporins (ceftazidime, cefotaxime, and ceftriaxone). Isolates demonstrating Extended-Spectrum activity are conjugated with susceptible E. Coli recipients to identify transmissible beta-lactam resistance genes Transconjugates are screened for the presence of TEM or SHV-type beta-lactamase genes by polymerase chain reaction and Southern blot hybridizations. Products obtained by PCR are sequenced to identify novel mutations. Clones harboring mutations are cloned and resequenced to confirm original findings. Data obtained from this proposal will be instrumental in (1) the early detection of mutant TEM and SHV-type beta-lactamases for proper treatment, (2) maximizing the use of antibiotics currently available, (3) establishing new forms of treatment, and (4) elucidating the trends and mechanisms of drug resistance and transmission of TEM and SHV-type beta-lactamases during nosocomial outbreaks. This project will involve students in every aspect. Students will gain invaluable knowledge and training in the fundamentals of DNA manipulations, PCR technology, analysis of clinical samples, DNA sequencing, data collection, and manuscript preparation. Training obtained from this study will prepare students for doctoral studies in cell, molecular, and microbiology.